This project focuses on the design and synthesis of peptide antigens, the immunological characterization of peptide hormone receptors, structure- function relationship studies, determination of amino acid sequences, and the identification and characterization of post-translational modifications of proteins: (1) We obtained high-level expression of a 92 residue protein, designated as LE93 corresponding to the C-terminal sequence (residues 268-359) of the angiotensin II (AII) receptor type 1b in E. coli strain TB1 transfected with pMAL-2c vector consisting of the inserted gene down stream of the malE maltose binding protein gene, and a protease Factor Xa site. The antiserum raised against this protein recognized a 66-67 kDa protein as the AII receptor with high specificity as judged from Western blot analyses of bovine adrenal plasma membrane and cells, and rat tissue homogenates, as well as immunoprecipitation of the radioiodinated [Sar-1,p-azidoPhe-8]AII photoaffinity labeled receptor from the same samples. (2) Nine peptides covering all putative cytoplasmic and extracellular domains of the murine GnRH receptor were synthesized, conjugated to bovine thyroglobulin, and the conjugates were used to raise antibodies in rabbits. (3) The gene encoding MAP30 (anti- HIV protein from Momordica charantia) has been cloned based on two sequences of tryptic fragments and used to express MAP 30 in E. coli using the expression vector pRSET. The recombinant protein exhibited identical anti-HIV and anti-tumor activities to those of the plant protein. (4) In addition to N-glycosidase activity on 28S ribosomal RNA and topoisomerase activity on plasmid and viral DNAs including HIV-1 long terminal repeats, MAP 30 and GAP 31 (anti-HIV protein from Gelonium multiflorum) were shown to inhibit 3'-processing, integration, and disintegration catalyzed by the HIV-1 integrase. These findings suggest that impediment of viral DNA integration may play a key role in the anti- HIV activity of these plant proteins. (5) A novel protein kinase (PK) C substrate, PP28 (Mr=28 kDa), has been purified from the heat- and acid- stable fraction of rat brain extract. Based on two segments of the N- terminal sequence, a cDNA encoding PP28 was cloned to express the protein in E. coli using the expression vector pRSET. It encodes a 180 amino acid sequence. Although both tissue and recombinant PP28 can be phosphorylated by PKA, PKC, and casein kinases(CK) I and II in vitro, only CK II appears to phosphorylate the protein in N1E115 neuroblastoma cells in vivo. The in vitro CK II phosphorylation of PP28 yielded phosphorylated Ser-62 and Ser-59 as predicted for CK II phosphorylation. The PP28 protein, which is widely distributed in tissues, may thus may be phosphorylated in a tissue-specific or in a highly regulated manner.